Báo cáo khoa học: Gain of structure and IgE epitopes by eukaryotic expression of the major Timothy grass pollen allergen, Phl p 1 pdf

Gain of structure and IgE epitopes by eukaryotic
expression of the major Timothy grass pollen allergen,
Phl p 1
Tanja Ball1,2, William Edstrom2, Ludwig Mauch3, Jacky Schmitt3, Bernd Leistler3, Helmut Fiebig4,
Wolfgang R. Sperr5, Alexander W. Hauswirth5, Peter Valent5, Dietrich Kraft1, Steven C. Almo2
and Rudolf Valenta1
1
2
3
4
5

Department of Pathophysiology, Center for Physiology and Pathophysiology, Medical University of Vienna, Austria
Albert Einstein College of Medicine, Department of Biochemistry, NY, USA
Pharmacia Diagnostics, Freiburg, Germany
Allergopharma KG, Reinbek, Germany
Division of Hematology, Department of Internal Medicine I, Medical University of Vienna, Austria

Keywords
allergen; allergy; epitope; eukaryotic
expression; Phl p 1
Correspondence
R. Valenta, Division of Immunopathology,
Department of Pathophysiology, Center for
Physiology and Pathophysiology, Medical
University of Vienna, Waehringer Guertel
18–20, A-1090 Vienna, Austria
Fax: +43 1 40 400 5130
Tel: +43 1 40 400 5108
E-mail:
(Received 6 August 2004, revised 21

September 2004, accepted 22 September
2004)
doi:10.1111/j.1432-1033.2004.04403.x

Approximately 400 million allergic patients are sensitized against group 1
grass pollen allergens, a family of highly cross-reactive allergens present in
all grass species. We report the eukaryotic expression of the group 1 allergen from Timothy grass, Phl p 1, in baculovirus-infected insect cells.
Domain elucidation by limited proteolysis and mass spectrometry of the
purified recombinant glycoprotein indicates that the C-terminal 40% of
Phl p 1, a major IgE-reactive segment, represents a stable domain. This
domain also exhibits a significant sequence identity of 43% with the family
of immunoglobulin domain-like group 2 ⁄ 3 grass pollen allergens. Circular
dichroism analysis demonstrates that insect cell-expressed rPhl p 1 is a
folded species with significant secondary structure. This material is well
˚
behaved and is adequate for the growth of crystals that diffract to 2.9 A
resolution. The importance of conformational epitopes for IgE recognition
of Phl p 1 is demonstrated by the superior IgE recognition of insect-cell
expressed Phl p 1 compared to Escherichia coli-expressed Phl p 1. Moreover, insect cell-expressed Phl p 1 induces potent histamine release and
leads to strong up-regulation of CD203c in basophils from grass pollen
allergic patients. Deglycosylated Phl p 1 frequently exhibits higher IgE
binding capacity than the recombinant glycoprotein suggesting that rather
the intact protein structure than carbohydrate moieties themselves are
important for IgE recognition of Phl p 1. This study emphasizes the
important contribution of conformational epitopes for the IgE recognition
of respiratory allergens and provides a paradigmatic tool for the structural
analysis of the IgE allergen interaction.

Type I allergy is an IgE-mediated hypersensitivity disease affecting more than 25% of the population [1,2].
Grass pollen allergens belong to the group of most frequently recognized allergenic components [3]. At least

40% of allergic patients are sensitized to grass pollen

allergens and more than 95% of them display IgE
reactivity to group I allergens [3–6]. Group 1 allergens
represent a family of glycoprotein allergens of
approximately 30 kDa that occur as cross-reactive
antigens in almost all grasses and corn species [6,7].

Abbreviations
PrPhl p 1, prokaryotic recombinant Phl p 1; ErPhl p 1, eukaryotic recombinant Phl p 1; GST, glutathione S-transferase.

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Eukaryotic expression of Phl p 1

They are exclusively expressed in mature pollen grains
where they are localized mainly in the cytoplasm [8].
Using immuoelectronmicroscopy two mechanisms for
the release of group 1 allergens have been demonstrated. First, contact of intact pollen grains with mucosal
surfaces (e.g. nasal epithelium) leads to hydration and
rapid diffusion of the allergens [9]. Second, it has been
demonstrated that rain water induces the expulsion of
respirable micron size allergen-containing particles
from grass pollens [10,11]. The small size of these subcellular particles allows them to reach the deeper airways and may explain the frequent occurrence of
heavy asthma attacks after rainfalls [12,13].
cDNAs coding for group 1 allergens from several
grasses have been isolated and showed high sequence

similarity [14–20]. The recombinant group 1 allergen
from Timothy grass, rPhl p 1, expressed in Escherichia
coli contained many of the T cell epitopes of natural
group 1 allergens and cross-reacted with the naturally
occuring isoallergens from Timothy grass and other
grass species [6,21,22]. However, several post-translational modifications (e.g. glycosylation, occurrence of
hydroxyprolines) and the formation of disulphide
bonds have been described for group I allergens
[23,24]. These modifications do not occur when proteins are expressed in prokaryotic expression systems
and hence E. coli-expressed group 1 allergens exhibit
impaired structural and immunological properties. The
importance of conformational epitopes for IgE recognition of group I allergens is highlighted by IgE competition experiments using recombinant fragments of
Phl p 1 representing continuous IgE epitopes as even
a mixture of several major IgE-epitope-containing
rPhl p 1 fragments does not completely inhibit IgE
binding to intact Phl p 1 [25].
To obtain properly folded rPhl p 1, the cDNA coding for the mature allergen was expressed in baculovirus-infected insect cells. An expression strategy was
chosen which led to the secretion of the recombinant
allergen into the cell culture supernatants. rPhl p 1
was purified to homogeneity, characterized by mass
spectro-metry and the presence of post-translational
modification (i.e. glycosylation) was investigated. The
secondary structure content of insect cell-expressed
rPhl p 1 was examined by circular dichroism analysis
and diffraction quality crystals of the recombinant
allergen were grown. The IgE binding properties of
insect cell-expressed Phl p 1 were compared with those
of E. coli-expressed and natural Phl p 1 by competition
studies performed under native conditions and the
importance of glycosylation for IgE reactivity was

examined by enzymatic deglycosylation of insect cellexpressed Phl p 1. Finally, the biological activity of
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T. Ball et al.

insect cell-expressed and E. coli-expressed Phl p 1 was
compared in histamine release experiments and by
CD203c expression in basophils from grass pollen
allergic patients [26]. The finding that proper folding
of insect-cell expressed Phl p 1 is related to increased
IgE reactivity and allergenic activity is discussed as a
general feature of respiratory allergens and has relevance for the development of allergy vaccines which
are based on the reduction of allergen fold.

Results
Comparison of natural and recombinant group 1
grass pollen allergens
Although the majority of rPhl p 1 was detected in the
insoluble pellet fraction of infected insect cells, up to
0.75 mgỈL)1 of soluble rPhl p 1 could be purified from
the culture supernatant by Ni2+-affinity chromatography under nondenaturing conditions. Purified insect
cell-expressed Phl p 1 migrated at slightly higher
molecular mass than the natural Phl p 1, E. coliexpressed Phl p 1 and the Phl p 1-homologous allergen
from rye grass (Lol p 1) (Fig. 1A). Insect cell-expressed
Phl p 1 as well as natural group 1 allergens (nPhl p 1
and nLol p 1) reacted with a rabbit antiserum raised
against purified E. coli-expressed Phl p 1 (Fig. 1B)
but not with the corresponding preimmune serum
(Fig. 1C). A band of approximately double the
molecular mass as the purified allergens, possibly representing a dimer, was detected in the bacterial and

insect cell-expressed Phl p 1 and, to a lower degree, in
the nPhl p 1 preparations.
Biochemical and biophysical characterization
of insect-cell expressed Phl p 1
The molecular mass of insect cell-expressed Phl p 1
was determined by mass spectrometry to be 28 122
Dalton (data not shown). The difference of 956 Da
between the calculated (27 166 Da) and the determined molecular mass is attributed to glycosylation.
Limited proteolysis in combination with mass spectrometry was performed to identify structural domains
[27,28]. Fundamental to this approach is the notion
that protection against proteolysis is conferred in
regions of the protein that are within a rigid structure, while proteolytic cleavage of a multiple-domain
protein is biased towards solvent accessible regions
(i.e. exposed loops, interdomain linker chains). We
identified two proteolytically stable structural domains
of rPhl p 1 by limited proteolysis, one comprising
C78–K118 and a second domain spanning from
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T. Ball et al.

Eukaryotic expression of Phl p 1

A

B

C

Fig. 1. Coomassie staining and immunoreactivity of purified recombinant and natural group 1 allergens. (A) Coomassie stained SDS ⁄ PAGE
containing natural Lol p 1 (nLol p 1), natural Phl p 1 (nPhl p 1), eukaryotic recombinant Phl p 1 (ErPhl p 1) and bacterial recombinant Phl p 1
(PrPhl p 1). B and C represent immunoblots probed with rabbit anti-(Phl p 1 Ig) antiserum and the corresponding preimmune serum, respectively.

K147–K241 (data not shown). The latter corresponds
to the region homologous to group 2 allergens. To
confirm that the difference between the calculated
and determined molecular mass is due to glycosylation of the insect cell-expressed Phl p 1, glycan detection was performed (Fig. 2A). Nitrocellulose-blotted
insect cell-expressed Phl p 1, but not E. coli-expressed
Phl p 1 showed a positive staining for glycan moieties
(blue color). Also, a nonglycosylated control protein,
creatinase, and the marker proteins gave negative
reaction in the glycan staining and appear brown
(Fig. 2A). Finally, enzymatic deglycosylation with
PNGase F resulted in a reduction of molecular mass
of insect cell-expressed Phl p 1 as visualized by
SDS ⁄ PAGE (Fig. 2B).
Insect cell-expressed Phl p 1 represents a folded
protein with considerable b-sheet structure that
crystallizes as thin plates
Insect cell- and E. coli-expressed Phl p 1 were analyzed
by circular dichroism (CD) spectroscopy to determine
their secondary structural content (Fig. 3). The CD
spectrum of insect cell-expressed, eukaryotic Phl p 1
(ErPhl p 1) suggested the presence of substantial antiparallel b-sheet, while the CD spectrum for the Phl p 1
expressed in bacteria (prokaryotic: PrPhl p 1) indicated
a considerable amount of unordered structure. The
characteristics of the CD spectrum of insect cellexpressed Phl p 1 indicates structural similarity with
Phl p 2, an almost exclusively b-sheet containing allergen with 43% sequence identity to the C-terminal third
of Phl p 1 [29,30]. Thermal denaturation of insect cellexpressed Phl p 1 was monitored by far-UV CD in the

range of 20 °C to 90° and showed an irreversible
unfolding transition, with a melting point of  42 °C
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(data not shown). Only the properly folded insect
cell-expressed but not the E. coli-expressed Phl p 1
afforded crystals. These crystals belonged to the orthorhombic space group P212121 and diffracted X-rays to
˚
a resolution of 2.9 A (Fig. 4).
Phl p 1 and Phl p 2 belong to different clusters of
proteins as determined by phylogenetic analysis
Amino acid sequences of seven group 1 pollen allergens and four group 2 ⁄ 3 allergens were subjected to
phylogenetic analysis using the phylip 3.6a2 package
( />(Fig. 5). The allergens formed three main clusters, one
comprising Zea m 1 and Cyn d 1, a second consisting
of Lol p 3, Dag g 3, Lol p 1, Phl p 1, Ory s 1 and
Tri a 3 and a third cluster including Lol p 2, Hol l 1,
Phl p 2 and Pha a 1. Although Phl p 1 and Phl p 2 are
derived from Phleum pratense and share high sequence
identity, the phylogenetic analysis shows that they are
less related to each other than group 1 and group 2 ⁄ 3
allergens from different species.
Insect cell-expressed Phl p 1 contains the IgE
epitopes of natural Phl p 1
A comparison of the IgE binding capacity of E. coliand insect cell-expressed Phl p 1 under nondenaturing
conditions in a dot-blot assay showed that insect cellexpressed Phl p 1 was more potent than the E. coliderived allergen (Table 1). IgE competition studies
performed under native conditions confirmed this
result (Fig. 6A). Preincubation of sera from four grass
pollen allergic patients with E. coli-expressed Phl p 1
completely inhibited IgE binding to the very same protein, but not to the insect cell-expressed Phl p 1. An

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Eukaryotic expression of Phl p 1

almost complete reduction of IgE binding to insect
cell-expressed Phl p 1 was only observed for serum of
patient 2, whereas considerable IgE reactivity of sera
1, 3 and 4 to insect cell-expressed Phl p 1 was observed
A

T. Ball et al.

despite preincubation with an excess of E. coliexpressed Phl p 1.
Whether insect cell-expressed Phl p 1 contains the
IgE epitopes of a natural Phl p 1 preparation was investigated by IgE competition experiments (Fig. 6B). Preincubation of sera from grass pollen allergic patients
with insect cell-expressed Phl p 1 led to a strong or
complete inhibition of IgE reactivity to natural Phl p 1
(Fig. 6B).
Next we studied the influence of glycosylation on
the IgE binding capacity of insect cell-expressed
Phl p 1 (Fig. 6C). Five out of 10 patients showed
stronger IgE reactivity to deglycosylated insect cellexpressed Phl p 1 than to the untreated protein
(Fig. 6C, 1, 2, 3, 5, 7). Three patients exhibited comparable IgE reactivity to both protein forms (Fig. 6C,
#4, 9, 10) and two sera reacted stronger with the glycosylated allergen version (Fig. 6C, 6, 8). Finally, we
studied the possible presence of cross-reactive IgE epitopes between Phl p 1 and Phl p 2. Preincubation of
sera from pollen allergic patients with insect cellexpressed, E. coli-expressed Phl p 1 or an unrelated
control allergen (birch pollen allergen, rBet v 1) had
no effect on IgE binding to rPhl p 2 (data not shown).
Allergenic activity of insect cell-expressed Phl p 1

B

The allergenic activity of insect cell-expressed Phl p 1
was analyzed by basophil histamine release (Fig. 7)
and CD203c expression (data not shown). Basophils
from two grass pollen allergic patients were exposed to
different concentrations of E. coli- or insect cellexpressed Phl p 1 (Fig. 7A,B). In both patients, insect
cell-expressed Phl p 1 was more potent, inducing histamine release at lower concentrations (10)3 lgỈmL)1)
than E. coli-expressed Phl p 1 (10)2 lgỈmL)1). Measurement of CD203c expression on blood basophils of
three Phl p 1 allergic patients confirmed these results.
Incubation with insect cell-expressed Phl p 1 always
led to stronger upregulation of CD203c than incubation with E. coli-expressed Phl p 1 (data not shown).

Fig. 2. Biochemical and biophysical characterization of insect cellexpressed Phl p 1. (A) Glycan detection. Nitrocellulose blotted
insect cell-expressed rPhl p 1 (ErPhl p 1), rPhl p 1 expressed in
bacteria (PrPhl p 1), Creatinase and marker proteins (M) were simultaneously stained for sugar moieties (blue) and reactive amino
groups (fluorescent). Molecular masses are indicated on the left
margin. (B) SDS ⁄ PAGE containing insect cell-expressed Phl p 1
before (ErPhl p 1-) and after (ErPhl p 1 +) enzymatic deglycosylation. Lane M: Molecular mass marker.

220

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Eukaryotic expression of Phl p 1

Fig. 3. Comparison of E. coli- and insect cell-expressed Phl p 1 by
circular dichroism spectroscopy. Far-UV CD spectra of E.coli- (grey)
and insect cell-expressed Phl p 1 (black), expressed as mean residue ellipticity (y-axis), were recorded at 20 °C in the wave length
range displayed on the x-axis.

Fig. 5. Analysis of the sequence and phylogenetic relationship
among group 1 and group 2 ⁄ 3 allergens from various grass species. A phylogenetic tree was reconstructed on the basis of aminoacid sequences of group 1 (Zea m 1: Zea mays, Cyn d 1: Cynodon
dactylon, Pha a 1: Phalaris aquatica, Hol l 1: Holcus lanatus,
Ory s 1: Oryza sativa, Lol p 1: Lolium perenne, Phl p 1: Phleum pratense) and group 2 ⁄ 3 allergens (Lol p 3: Lolium perenne, Dag g 3:
Dactylis glomerata, Lol p 2: Lolium perenne, Tri a 3: Triticus aestivum, Phl p 2: Phleum pratense) using the PROTDIST and KITSCH
program of the PHYLIP package.
Fig. 4. Crystal growth of insect cell-expressed Phl p 1.Phl p 1 crystallizes as thin plates of 0.35 · 0.35 · 0.15 mm.

Discussion
Phl p 1 represents one of the most important respiratory
allergens known to date. As Phl p 1 is a glycoprotein
containing seven cysteines, we expressed the allergen in
eukaryotic insect cells to obtain a post-translationally
modified and folded protein. As demonstrated by mass
spectrometry, glycan detection and deglycosylation
experiments, insect cell-expressed Phl p 1 was obtained
as a glycoprotein. The seemingly correct folding of
insect cell-expressed Phl p 1 is demonstrated by the following experiments: insect cell-expressed Phl p 1 but
not E. coli-expressed Phl p 1 exhibited a secondary
structure consisting mainly of b-sheets when analyzed
by CD spectroscopy. Furthermore, only insect
cell-expressed Phl p 1 grew diffraction quality crystals
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and thus will yield the three-dimensional structure of

this allergen.
Phl p 1 belongs to the group 1 family of highly crossreactive grass pollen allergens. The C-terminal domains
of these allergens display sequence similarity to group
Table 1. Comparison of the IgE binding capacity of E. coli- and
insect cell-expressed Phl p 1. IgE reactivity to recombinant Phl p 1,
expressed in E. coli (PrPhl p 1) and baculovirus-infected insect cells
(ErPhl p 1).
PrPhl p 1

ErPhl p 1

Patient
number

IgE binding (c.p.m.)

IgE binding (c.p.m.)

1
2
3
4
5
6

158
4544
724
1010
963

1193

865
6370
2309
2980
3918
3889

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Eukaryotic expression of Phl p 1

T. Ball et al.

A
B

C

Fig. 6. (A) Superior IgE binding capacity of insect cell- vs. E. coli-expressed rPhl p 1. Sera from four grass pollen allergic patients were preincubated with bacterially expressed rPhl p 1 and exposed to dot-blotted bacterial recombinant (PrPhl p 1+) or eukaryotic recombinant Phl p 1
(ErPhl p 1+). PrPhl p 1- and ErPhl p 1- show the IgE binding without preadsorption of sera. (B) Inhibition of IgE binding to natural Phl p 1
(nPhl p 1) by insect cell-expressed Phl p 1. Sera from three grass pollen allergic patients (1–3) were tested for IgE reactivity to nitrocellulosedotted eukaryotic recombinant Phl p 1 (ErPhl p 1) and natural Phl p 1 (nPhl p 1). Sera were preadsorbed with BSA (A), natural Phl p 1 (B), or
insect cell-expressed Phl p 1 (C). (C) IgE binding capacity of deglycosylated insect cell-expressed Phl p 1.IgE reactivity of 10 sera from grass
pollen allergic patients (1–10) to untreated (–) and deglycosylated (+) Phl p 1 is shown.

2 ⁄ 3 grass pollen allergens, another family of major grass
pollen allergens that exhibit an immunoglobulin-like
fold composed almost exclusively of b-sheet structure

[29,30]. As shown by circular dichroism spectroscopy,
Phl p 1 showed also almost exclusively b-sheet secondary structure. The results from limited proteolysis combined with mass spectrometry indicated a two domain
organization of the protein with a C-terminal portion
homologous to group 2 allergens. Despite these findings,
Phl p 1 and Phl p 2 appear to represent immunologically independent allergens because significant crossreactivity of IgE antibodies was not observed and both
proteins belonged to different phylogenetic clusters.
The analysis of Phl p 1 IgE epitopes using recombinant allergen fragments had indicated the presence
222

of several continuous IgE epitopes, of which the most
prominent could be allocated to the C-terminal portion of Phl p 1 [25]. We have identified this portion
as an intact domain by the limited proteolysis experiment suggesting that intact and folded Phl p 1
domains represent the primary targets for patients’
IgE antibodies. The latter assumption is also supported by the fact that only an incomplete inhibition of
IgE reactivity to the Phl p 1 allergen could be
obtained after preincubation of patients’ sera with
small recombinant protein fragments suggesting the
importance of conformational IgE epitopes [25].
Therefore, we further tested the importance of structural integrity on IgE binding capacity and allergenic
activity of Phl p 1 by comparing insect cell-expressed

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T. Ball et al.

Fig. 7. Induction of basophil histamine release with recombinant
Phl p 1 preparations. Granulocytes from patients (A, B) allergic to
grass pollen were incubated with various concentrations (x-axis) of
bacterial rPhl p 1 (PrPhl p 1) and eukaryotic rPhl p 1. The percentage

of histamine released into the supernatant is displayed on the y-axis.

Phl p 1 with E. coli-expressed Phl p 1. This comparison revealed a higher IgE-binding capacity and more
pronounced allergenic activity of insect cell-expressed
rPhl p 1 compared to E. coli-expressed rPhl p 1, as
determined by basophil activation assays. Deglycosylation experiments demonstrate that the higher IgEbinding capacity and increased allergenic activity of
insect cell-expressed Phl p 1 is due to intact structural
integrity rather than to IgE recognition of carbohydrate moieties. In fact, we found that deglycosylation rather increased the IgE binding capacity of
Phl p 1. This may be due to the exposure of protein
epitopes by removing carbohydrates from a potentially hyperglycosylated insect cell-expressed Phl p 1.
On the other hand, it is unlikely that the authentic,
plant-derived carbohydrates represent per se important targets for patients’ IgE antibodies because insect
cell-expressed Phl p 1 showed almost identical IgE
reactivity as natural Phl p 1.
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Eukaryotic expression of Phl p 1

The importance of native tertiary structure for the
IgE recognition of Phl p 1 seems to be a general principle applicable to the most common respiratory allergens. For example, it has been demonstrated that
disruption of native structure by fragmentation has led
to a strong reduction of the IgE binding capacity and
allergenic activity of the major birch pollen allergen,
Bet v 1 [31], the cross-reactive calcium-binding allergens
Aln g 4 [32] and Phl p 7 [33], the major mite allergen
Der p 2 [34], and the major bovine allergen, Bos d 2
[35]. We consider the possibility that respiratory allergens may predominantely contain conformational IgE
epitopes as important for at least three reasons. First, it
indicates that respiratory sensitization occurs preferentially against intact and folded protein antigens which
elute from respirable particles (e.g. pollen, mite faeces,

animal dander). Second, our study emphasizes that it is
important to choose an optimal expression strategy for
obtaining native properly folded recombinant allergens
which closely mimic the immunological properties of the
natural counterparts for diagnostic purposes.
Finally, and perhaps most importantly, IgE recognition of mainly conformational epitopes has important
implications for the design of safe allergy vaccines with
reduced allergenic activity. Disruption of the native
structure of respiratory allergens allows for the maintenance of important T cell epitopes of a given allergen
and simultaneously preserves sequences relevant for the
induction of protective antibody responses [36]. Controlled reduction of the fold of respiratory allergens by
recombinant DNA technology or synthetic peptide
chemistry thus seems to be a generally applicable strategy for the generation of recombinant allergy vaccines
with reduced allergenic activity [37].

Experimental procedures
Materials, patients’ sera and antibodies
The Sf9 cell line was purchased from the German Collection of Microorganisms and Cell Cultures (Braunschweig,
Germany). After informed consent was obtained, sera were
collected from Phl p 1 allergic patients, following the
Helsinki guidelines. Allergenic patients were characterized
by case history, skin prick test, and the demonstration of
allergen-specific serum IgE antibodies by RAST (Pharmacia
Diagnostics, Uppsala, Sweden). Natural group 1 grass pollen
allergens from Timothy grass (nPhl p 1) and rye grass
(nLol p 1) were purified as described [38]. Purified
E. coli-expressed rPhl p 1, rPhl p 2 and rBet v 1 were
obtained from BIOMAY (Vienna, Austria). A rabbit antiPhl p 1 antiserum was obtained by immunizing rabbits with
purified rPhl p 1 using complete Freunds’ adjuvant (Charles

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Eukaryotic expression of Phl p 1

River, Kissleg, Germany). Alkaline phosphatase-conjugated
goat anti-(rabbit Ig) and rabbit anti-(mouse Ig) serum was purchased from JacksonImmunoResearch Laboratories (West
Grove, PA, USA), a mouse monoclonal anti-Hexahistidine
antibody was obtained from Dianova (Hamburg, Germany).
The 125I-labeled anti-human IgE immunoglobulins were purchased from Pharmacia Diagnostics.

Construction of recombinant baculovirus
The Phl p 1-encoding cDNA [16] was PCR amplified and
cloned into the BamHI and KpnI restriction sites of the
pBacPAK8 vector (Clontech Inc., Palo Alto, CA, USA),
containing the baculovirus-derived ecdysteroid UDPglucosyltransferase signal peptide [39] for enhanced secretion of
the recombinant protein into the culture supernatant and a
C-terminal His6-tag. The pBacPAK8 construct was confirmed by DNA sequencing and cotransfected with the linearized pBacPAK6 viral DNA (Clontech Inc., Palo Alto,
CA, USA) into Sf9 insect cells. The clones with the highest
level of protein secretion were chosen by Western blotting
for virus amplification.

Expression and purification of rPhl p 1 from
baculovirus-infected insect cells
The expression of rPhl p 1 in insect cells was optimized by
infecting Sf9 cells with different amounts of virus and by
expression for various periods. Aliquots of the culture supernatants and cell pellets were analyzed by SDS ⁄ PAGE and
immunoblotting with a rabbit anti-Phl p 1 antiserum and
a monoclonal anti-hexahistidine antibody. Rabbit antirPhl p 1 Igs were detected with an alkaline phosphatase
(AP)-labeled goat anti-(rabbit Ig) antiserum. Bound antihexahistidine Igs were detected with AP-labeled rabbit antimouse Igs.

Optimal expression of Phl p 1 was achieved by infection
of 2 · 106 Sf9 cells per mL with recombinant baculovirus
at a multiplicity of infection (MOI) of 5 with culturing in
3 L spinner ⁄ flasks in Insect-Xpress medium (BioWhittaker
Inc., Walkersville, MD, USA) containing 2% fetal bovine
serum. At day two postinfection, supernatants were separated by centrifugation (8000 g, 4 °C, 30 min) and dialyzed
against start buffer [50 mm sodium phosphate (pH 8.0),
300 mm NaCl] at 4 °C overnight. Insect cell-expressed
rPhl p 1 was purified using Ni-nitrilotriacetic acid superflow
matrix (Qiagen, Hilden, Germany) under nondenaturing
conditions by stepwise elution with increasing (20–250 mm)
imidazole concentrations. The eluted samples were dialyzed
against 10 mm Tris HCl (pH 8.0), 100 mm NaCl and concentrated by Centricon ultrafiltration (Millipore, Bedford,
MA, USA). Protein concentrations of purified samples were
estimated using BCA reagent (Pierce Chemicals, Rockford,
IL, USA) and UV absorption at 280 nm. The molar extinc-

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T. Ball et al.

tion coefficient of the protein was calculated from the tyrosine and tryptophan content [40].

Mass spectrometry
Purified baculovirus-expressed Phl p 1 was analyzed by
LC-MS (Liquid Chromatography-Mass Spectrometry)
using a VYDAC (Hesperia, CA, USA) C4 column on a
Waters HPLC 2690 (Waters Corp., Milford, MA, USA)
which fed into an electrospray Thermo Finnigan LCQ
quadrupole ion-trap mass spectrometer (ThermoQuest Inc.,

San Jose, CA, USA).

Limited proteolysis followed by LC-MS
Purified baculovirus-expressed Phl p 1 was subjected to limited proteolysis by trypsin, Arg-C, Lys-C, Asp-N and Glu-C.
Ten microliter aliquots containing 18 lm rPhl p 1 were digested with protease in the following ratios 1 : 5, 1 : 15, 1 : 50,
1 : 150 and 1 : 500 (protease:rPhl p 1; w ⁄ w) for 1 h at room
temperature. Proteolysis was halted by freezing at )70 °C.
Aliquots were analyzed by SDS ⁄ PAGE. Samples showing
multiple bands, indicative for successful partial digest were
then selected for further investigation by LC-MS. A Vydac
C18 column was used on a Waters HPLC 2690 (Waters
Corp.) followed by electrospray into a Thermo Finnigan
LCQ Ion Trap Mass Spectrometer (ThermoQuest Inc.). The
spectra were deconvoluted using Thermo Finnigan’s xcalibur software and the spectra were also verified by hand calculations of charge states. The proteolytic fragments were
identified using the paws software program (version 8.1.1, for
Macintosh; Genomic SolutionsTM , Ann Arbor, MI, USA;
/>
Detection of glycoproteins and deglycosylation
treatment
Purified E. coli- and insect cell-expressed Phl p 1 proteins
were separated by SDS ⁄ PAGE and transferred to nitrocellulose followed by detection of sugars using a DIG glycan ⁄ protein double labeling kit (Boehringer Mannheim GmbH,
Mannheim, Germany). Briefly, glycans were oxidized to produce aldehyde groups allowing the covalent attachment of
the steroid hapten digoxigenin (DIG). The latter was then
detected using horseradish peroxidase-conjugated anti-digoxigenin Igs yielding a blue color reaction. Creatinase and
bacterial rPhl p 1 were used as nonglycosylated controls
which were stained by labeling of amino groups with fluorescein and detection with alkaline phosphatase-conjugated
anti-fluorescein Igs (Boehringer) giving a brown color reaction. Enzymatic deglycosylation was performed with glutathione S-transferase (GST)–PNGase F (Hampton Research,
CA, USA) by using reaction ratios of GST–PNGase F:glycoprotein of 1 : 2. Deglycosylation was carried out for 15 h at

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T. Ball et al.

room temperature in 10 mm Tris (pH ¼ 8.0), 100 mm NaCl.
The GST–PNGase F was removed from the target protein
using glutathione-Sepharose.

Circular dichroism (CD) measurements
All recombinant proteins were subjected to CD analysis to
access stability and secondary structure composition. Far
UV-CD spectra were collected on a Jasco-J720 spectropolarimeter (Jasco, Tokyo, Japan) at room temperature, at final
protein concentrations of 10–25 lm in either 0.5 or
0.01 mm path-length quartz cuvettes. The molar ellipticity
was calculated according to [h] ¼ ⁄ 10cl, where h is the ellipticity, l is the cuvette path-length in cm and c is the protein
concentration in molỈL)1. Three independent measurements
were recorded and averaged for each spectral point in all
experiments. Thermal denaturation was monitored in the
range of 20 °C to 90 °C. The reversibility of the unfolding
process was checked by measuring the CD signal upon
cooling to the starting temperature.

Phylogenetic analysis of the relationships among
group 1 and group 2/3 allergens from various
grass species
Multiple alignment of sequences homologous to Phl p 1 as
identified by a BLAST search [41] was generated by using
clustalx [42]. A distance matrix among sequences was
constructed using the protdist program of the phylip
3.6a2 [43,44] package. The distance matrix was used as

input to the KITSCH program from the phylip package
for the construction of a phylogenetic tree. This program
implements the Fitch–Margoliash least-squares methods
with the assumption of an evolutionary clock.

SDS/PAGE analysis and immunoblotting
Samples were resolved on 12.5% polyacrylamide gels under
reducing conditions. Proteins were stained with Coomassie
blue or transferred to nitrocellulose membranes [45]. Blotted proteins were probed with sera from Phl p 1 allergic
patients¢, anti-His Igs, rabbit anti-Phl p 1 antiserum and
the corresponding preimmune serum. Patients’ bound IgE
antibodies were detected with 125I-labeled anti-human IgE
[46], anti-His Igs with AP-conjugated rabbit anti-(mouse
Ig), and bound rabbit Igs with an AP-conjugated goat anti(rabbit IgG) serum [47].

IgE-binding capacity and cross-reactivity
of allergens as determined by nondenaturing
dot-blot experiments
The IgE reactivity of the recombinant Phl p 1 molecules
was determined by dot-blot under conditions of antigen

FEBS Journal 272 (2005) 217–227 ª 2004 FEBS

Eukaryotic expression of Phl p 1

excess [7]. Three micrograms of the purified recombinant
proteins were dotted onto nitrocellulose strips and incubated with sera from Phl p 1 allergic patients. Bound IgE
antibodies were detected with 125I-labeled anti-(human IgE)
Igs (Pharmacia) and quantified by c-counting (Wallac,
LKB, Turku, Finland) [25].

IgE inhibition experiments under conditions of antigen
excess were performed as described [7]. Patients’ sera were
incubated with 5 lgỈmL)1 of each allergen (or the same
amount of BSA for control purposes) overnight at 4 °C.
The next day, preincubated sera were exposed to 3 lg of
nitrocellulose-dotted natural Phl p 1, rPhl p 2, E. coli and
baculovirus expressed Phl p 1. Bound serum IgE was detected as described for the IgE immunoblotting and quantified
by c-counting [25].

Basophil activation experiments
Granulocytes were isolated from heparinized blood samples
of individuals allergic to Phl p 1 by dextran sedimentation.
The capacity of E. coli- and insect cell-expressed Phl p 1 to
induce basophil degranulation was tested by incubation of
granulocytes with various concentrations of the purified
proteins and by measuring histamine released into the
cell-free supernatant by radioimmunoassay (Immunotech,
Marseille, France). Histamine release was measured in triplicates and expressed as a percentage of total histamine
determined after cell lysis, as described [48]. Up-regulation
of CD203c expression on basophils after allergen exposure
was measured as described [26].

Acknowledgements
We acknowledge the skillful technical assistance of
Miriam Gulotta regarding the circular dichroism
experiments. This study was supported by grants
Y078GEN, F01801, F01809, J1835 and J2122 of the
Austrian Science Fund, by the CeMM Project of the
Austrian Academy of Sciences, and by a research grant
from BIOMAY, Vienna, Austria.

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